The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.     

Escherichia coli signal recognition particle receptor FtsY contains an essential and autonomous membrane-binding amphipathic helix

Escherichia coli membrane protein biogenesis is mediated by a signal recognition particle and its membrane-associated receptor (FtsY). Although crucial for its function, it is still not clear how FtsY interacts with the membrane. Analysis of the structure/function differences between severely truncated active (NG+1) and inactive (NG) mutants of FtsY enabled us to identify an essential membrane-interacting determinant. Comparison of the three-dimensional structures of the mutants, combined with site-directed mutagenesis, modeling, and liposome-binding assays, revealed that FtsY contains a conserved autonomous lipid-binding amphipathic alpha-helix at the N-terminal end of the N domain. Deletion experiments showed that this helix is essential for FtsY function in vivo, thus offering, for the first time, clear evidence for the functionally important, physiologically relevant interaction of FtsY with lipids.     

GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP- dependent reaction during protein translocation is the SRP receptor- mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.     

Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites

Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRbeta subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY-SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.     

New prospects in studying the bacterial signal recognition particle pathway

In vivo and in vitro studies have suggested that the bacterial version of the mammalian signal recognition particle (SRP) system plays an essential and selective role in protein biogenesis. The bacterial SRP system consists of at least two proteins and an RNA molecule (termed Ffh, FtsY and 4.5S RNA, respectively, in Escherichia coli). Recent evidence suggests that other putative bacterial-specific SRP components may also exist. In vitro experiments confirmed the expected basic features of the bacterial SRP system by demonstrating interactions among the SRP components themselves, between them and ribosomes, ribosome-linked hydrophobic nascent polypeptides or inner membranes. The availability of a conserved (and essential) bacterial SRP version has facilitated the implementation of powerful genetic and biochemical approaches for studying the cascade of events during the SRP-mediated targeting process in vivo and in vitro as well as the three-dimensional structures and the properties of each SRP component and complex.     

信号识别颗粒(SRP)在膜蛋白定位中的作用

依赖信号识别颗粒(signal recognition particle,SRP)的共翻译转运是所有生命体中的一个保守途径,它将新生肽链的翻译与转运耦联在一起。超过30%的新合成的多肽链被SRP转运到正确位置。最近的研究表明,大肠杆菌中SRP抑制子可以规避SRP的需求。当SRP缺失时,翻译控制在介导膜蛋白定位方面起着关键作用。本综述总结了SRP底物如何在存在或缺失SRP的情况下转运到适当的位置以及翻译速率降低如何补偿SRP的缺失。我们还讨论了不同蛋白质对SRP的依赖程度。这一回顾将为进一步研究SRP功能及膜蛋白定位提供新思路。     

In vivo analysis of an essential archaeal signal recognition particle in its native host

The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.     

Export of beta-lactamase is independent of the signal recognition particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.     

The Streptomyces lividans cytoplasmic signal recognition particle receptor FtsY is involved in protein secretion

The bacterial version of the mammalian signal recognition particle (SRP) and its receptor alpha-subunit (FtsY) is well conserved and essential to all known bacteria. In gram-negative bacteria, the SRP pathway mediates a co-translational targeting of most inner membrane proteins. Additionally, in Streptomyces lividans, a gram-positive bacterium, SRP also targets secretory proteins to the translocon. The role of S. lividans FtsY has been assessed in this work. Co-immunoprecipitation studies confirmed that FtsY is associated with the S. lividans SRP in the cytoplasm and that this complex also co-immunoprecipitated with pre-agarase, suggesting that the SRP receptor is involved in SRP-mediated targeting of secretory proteins in S. lividans. Furthermore, the SRP remains attached for the most part to the cellular membrane when the cleavage of pre-secretory proteins is severely reduced in a strain lacking the gene coding for the major type-I signal peptidase.     

The signal recognition particle receptor is a complex that contains two distinct polypeptide chains

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.     

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.     

SecA is required for the insertion of inner membrane proteins targeted by the Escherichia coli signal recognition particle

Recent work has demonstrated that the signal recognition particle (SRP) is required for the efficient insertion of many proteins into the Escherichia coli inner membrane (IM). Based on an analogy to eukaryotic SRP, it is likely that bacterial SRP binds to inner membrane proteins (IMPs) co-translationally and then targets them to protein transport channels ("translocons"). Here we present evidence that SecA, which has previously been shown to facilitate the export of proteins targeted in a post-translational fashion, is also required for the membrane insertion of proteins targeted by SRP. The introduction of SecA mutations into strains that have modest SRP deficiencies produced a synthetic lethal effect, suggesting that SecA and SRP might function in the same biochemical pathway. Consistent with this explanation, depletion of SecA by inactivating a temperature-sensitive amber suppressor in a secAam strain completely blocked the membrane insertion of AcrB, a protein that is targeted by SRP. In the absence of substantial SecA, pulse-labeled AcrB was retained in the cytoplasm even after a prolonged chase period and was eventually degraded. Although protein export was also severely impaired by SecA depletion, the observation that more than 20% of the OmpA molecules were translocated properly showed that translocons were still active. Taken together, these results imply that SecA plays a much broader role in the transport of proteins across the E. coli IM than has been previously recognized.     

The signal recognition particle receptor alpha subunit assembles co-translationally on the endoplasmic reticulum membrane during an mRNA-encoded translation pause in vitro.

Many proteins, including the alpha subunit of the signal recognition particle receptor (SR alpha), are targeted within the cell by poorly defined mechanisms. A 140 residue N-terminal domain of SR alpha targets and anchors the polypeptide to the endoplasmic reticulum membrane by a mechanism independent of the pathway involving the signal recognition particle. To investigate the mechanism of membrane anchoring, translation pause sites on the SR alpha mRNA were used to examine the targeting of translation intermediates. A strong pause site at nucleotide 507 of the mRNA open reading frame corresponded with the shortest nascent SR alpha polypeptide able to assemble on membranes. An mRNA sequence at this pause site that resembles a class of viral -1 frameshift sequences caused translation pausing when transferred into another mRNA context. Site-directed mutagenesis of the mRNA greatly reduced translation pausing without altering the polypeptide sequence, demonstrating unambiguously a role for this mRNA sequence in translation pausing. SR alpha polypeptides synthesized from the non-pausing mRNA were impaired in co-translational membrane anchoring. Furthermore, co-translational membrane assembly of SR alpha appears to anchor polysomes translating SR alpha to membranes.     

The beta-subunit of the signal recognition particle receptor is a novel GTP-binding protein without intrinsic GTPase activity

The beta-subunit of the signal recognition particle receptor (SRbeta), a member of the Ras family of small molecular weight GTPases, is involved in the targeting of nascent polypeptide chains to the protein translocation machinery in the endoplasmic reticulum membrane. We purified SRbeta from an expressing strain of Escherichia coli and investigated the properties of the isolated GTPase. We find that, unlike other Ras family GTPases, most SRbeta purifies bound to GTP, and SRbeta-bound GTP is not easily exchanged with solution GTP. SRbeta possesses no detectable GTPase activity. Although a stable interaction between SRbeta and ribosomes is observed, SRbeta is not stimulated to hydrolyze GTP when incubated with ribosomes or ribosome-nascent chains. A GTPase mutant harboring a mutation in a region predicted to be functionally important, based on observations made in related GTPases, binds GTP with faster kinetics and appears to be a less stable protein but otherwise displays similar properties to the wild-type SRbeta GTPase. Our results demonstrate that as an isolated GTPase, SRbeta functions differently from the Arf- and Ras-type GTPases that it is most closely related to by sequence.     

X-ray structures of the signal recognition particle receptor reveal targeting cycle intermediates

The signal recognition particle (SRP) and its conjugate receptor (SR) mediate cotranslational targeting of a subclass of proteins destined for secretion to the endoplasmic reticulum membrane in eukaryotes or to the plasma membrane in prokaryotes. Conserved active site residues in the GTPase domains of both SRP and SR mediate discrete conformational changes during formation and dissociation of the SRP.SR complex. Here, we describe structures of the prokaryotic SR, FtsY, as an apo protein and in two different complexes with a non-hydrolysable GTP analog (GMPPNP). These structures reveal intermediate conformations of FtsY containing GMPPNP and explain how the conserved active site residues position the nucleotide into a non-catalytic conformation. The basis for the lower specificity of binding of nucleotide in FtsY prior to heterodimerization with the SRP conjugate Ffh is also shown. We propose that these structural changes represent discrete conformational states assumed by FtsY during targeting complex formation and dissociation.     

Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins

We have investigated the role of signal recognition particle (SRP) in the biosynthesis of Sindbis glycoproteins by translating the viral 26S mRNA in a wheat-germ cell-free system. SRP was shown to have no effect on the synthesis or proteolytic processing of the cytoplasmic C protein. In contrast, the membrane integration and the proteolytic processing of the viral glycoproteins PE2 and E1 were demonstrated to be SRP-dependent. In the absence of microsomal membranes, SRP caused an arrest of the synthesis of the viral glycoproteins. This arrest could be released by the addition of salt-extracted microsomal membranes. Synchronization experiments indicated that the uncleaved signal sequence of PE2 was recognized by SRP after at most 130 amino acids of PE2 had been polymerized. No apparent interaction of SRP with a putative signal sequence of E1 and/or a 6-kDa peptide could be detected.     

Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY

The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG's GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.     

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

Translocation of a lysosomal enzyme across the microsomal membrane requires signal recognition particle

Signal recognition particle (SRP), an 11S ribonucleoprotein (Walter and Blobel (1982) Nature 299, 691-698), is required for translocation of secretory proteins across microsomal membranes (Walter and Blobel (1980) Proc. Natl. Acad. Sci. USA 77, 7112-7116) and for asymmetric integration into microsomal membranes of a transmembrane protein (Anderson et al., (1982) J. Cell Biol. 93, 501-506). We demonstrate here that SRP is also required for translocation of the lysosomal protease cathepsin D across microsomal membranes.